Method for improving affinity of antibody for antigen and use thereof

ABSTRACT

Disclosed is a method for improving affinity of an antibody for an antigen, comprising, in an unmodified antibody, improving affinity for an antigen as compared to the unmodified antibody, by changing 17th, 18th and 20th amino acid residues of a light chain defined by Kabat method to charged amino acid residues.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority from prior Japanese Patent ApplicationNo. 2020-093411, filed on May 28, 2020, and Japanese Patent ApplicationNo. 2020-10411, filed on Jan. 24, 2020, entitled “METHOD FOR IMPROVINGAFFINITY OF ANTIBODY FOR ANTIGEN AND USE THEREOF”, the entire contentsof which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a method for improving affinity of anantibody for an antigen. The present invention relates to a method forproducing an antibody with improved affinity for an antigen as comparedto an unmodified antibody. The present invention relates to a modifiedantibody with improved affinity for an antigen as compared to anunmodified antibody.

BACKGROUND

Conventionally, a technique for changing affinity of an antibody for anantigen by modifying an amino acid sequence of the antibody has beenknown. For example, U.S. Patent Application Publication No. 2018/0179298describes a method for controlling affinity for an antigen by changingat least 3 amino acid residues in framework region 3 of an antibody tocharged amino acid residues. In this method, the affinity of theantibody for an antigen is improved while maintaining an amino acidsequence of complementarity determining region (CDR).

An object of the present invention is to provide a novel method forimproving affinity of the antibody for an antigen by modifying aminoacid residues of a framework region (FR) without modifying the aminoacid sequence of CDR, and a novel antibody with improved affinity for anantigen.

SUMMARY OF THE INVENTION

The scope of the present invention is defined solely by the appendedclaims, and is not affected to any degree by the statements within thissummary.

The present inventors have found that total value of amino acidfrequencies of predetermined amino acid residues at each position of anamino acid sequence of FR of a light chain of an antibody and a ratio ofsolvent-exposed surface area of each amino acid residue in the FR areinvolved in affinity of the antibody for an antigen, thereby completingthe present invention.

The present invention provides a method for improving affinity of anantibody for an antigen, comprising: by changing 17th, 18th and 20thamino acid residues of a light chain in unmodified antibody, thepositions of the amino acid residues being defined by Kabat method, tocharged amino acid residues, improving affinity for an antigen ascompared to the unmodified antibody.

The present invention provides a method for producing an antibody withimproved affinity for an antigen as compared to an unmodified antibody,including changing at least 3 amino acid residues selected from a groupconsisting of 17th, 18th and 20th amino acid residues of a light chainin the unmodified antibody, the positions of the amino acid residuesbeing defined by Kabat method, to charged amino acid residues, andrecovering the antibody obtained in the changing.

The present invention provides a modified antibody with improvedaffinity for an antigen as compared to an unmodified antibody. In thismodified antibody, at least 3 amino acid residues selected from a groupconsisting of 17th, 18th and 20th amino acid residues of a light chaindefined by Kabat method in the unmodified antibody are changed tocharged amino acid residues.

In certain embodiments, the charged amino acid residue is a basic aminoacid residue.

In certain embodiments, the antibody is an antibody fragment.

In certain embodiments, the antibody fragment is a Fab fragment, aF(ab′)2 fragment, a Fab′ fragment, a Fd fragment, a Fv fragment, a dAbfragment, scFv, or rIgG.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of an amino acid sequence analysis system;

FIG. 2 is a diagram showing a hardware configuration of an amino acidsequence analysis system;

FIG. 3 is a flowchart showing an analysis process of amino acid sequenceof an unmodified antibody by an analysis device of amino acid sequence;

FIG. 4 is a diagram showing an example of a display screen of analysisresults;

FIG. 5 is a flowchart showing an analysis process of amino acid sequencefor specifying candidates for antibody modification sites by theanalysis device of amino acid sequence;

FIG. 6 is a diagram showing an example of a display screen of analysisresults;

FIG. 7 is a flowchart showing an analysis process of amino acid sequencefor creating a sequence when candidates for antibody modification sitesare changed to charged amino acid residues by the analysis device ofamino acid sequence;

FIG. 8 is a flowchart showing an analysis process of amino acid sequencefor creating a sequence when candidates for antibody modification sitesare changed to basic amino acid residues when electrical characteristicof CDR is neutral by the analysis device of amino acid sequence; and

FIG. 9 is a photograph of a gel in which an antibody eluate wasseparated by SDS-PAGE and stained with Coomassie Brilliant Blue (CBB).

FIG. 10A is a light chain of wild-type humanized anti-HER2 antibody.

FIG. 10B is a heavy chain of wild-type humanized anti-HER2 antibody.

FIG. 10C is a heavy chain of Fab fragment of wild-type humanizedanti-HER2 antibody.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS [1. Method forImproving Affinity of Antibody for Antigen]

In the method for improving affinity of an antibody for an antigen ofthe present embodiment (hereinafter, also referred to as “method forimproving affinity”), among amino acid residues satisfying predeterminedconditions in an amino acid sequence of FR of a light chain of anantibody, at least 3 amino acid residues are changed to charged aminoacid residues. As a result, the affinity of the antibody for an antigenis improved as compared to that of an unmodified antibody. Here, anoriginal antibody whose affinity for an antigen is improved by themethod provided by the present disclosure described above is referred toas an “unmodified antibody”. As used herein, the term “unmodifiedantibody” means an antibody having an amino acid sequence beforeapplying the “method for improving affinity”. The “unmodified antibody”includes not only an antibody having a natural amino acid sequence(wild-type antibody) but also an antibody in which the amino acidsequence is artificially changed based on a method other than the“method for improving affinity”.

In the present embodiment, the unmodified antibody is not particularlylimited. Since it is not necessary to change the amino acid sequence ofCDR in the method for improving affinity of the present embodiment, anantibody that recognizes any antigen may be used. In a preferredembodiment, the unmodified antibody is an antibody in which a basesequence of a gene encoding a variable region of light chain is known oran antibody in which the base sequence can be confirmed. Specifically,it is an antibody in which a base sequence of an antibody gene isdisclosed in a known database, or an antibody in which a hybridomaproducing the antibody is available. Examples of the database includeGenBank, abYsis, IMGT, and the like. The antibody class may be IgG, IgA,IgM, IgD or IgE, and is preferably IgG. The unmodified antibody may bein a form of an antibody fragment as long as it has a variable regionincluding FR. Examples of the antibody fragment include Fab fragments,F(ab′)2 fragments, Fab′ fragments, Fd fragments, Fv fragments, dAbfragments, single chain antibodies (scFv), reduced IgG (rIgG), and thelike. Among them, Fab fragments are particularly preferred.

As an example of the unmodified antibody, amino acid sequences of alight chain and a heavy chain of a humanized anti-HER2 antibody(trastuzumab) and an amino acid sequence of a heavy chain of a Fabfragment are shown in FIGS. 10A-10C, respectively. In FIGS. 10A-10C, theunderlined parts indicate a variable region, and the gray marker partsindicate CDRs.

The amino acid sequences of each CDR and variable region of the lightchain of the wild-type humanized anti-HER2 antibody are as follows.

Light chain CDR1: (SEQ ID NO: 38) RASQDVNTAVA Light chain CDR2:(SEQ ID NO: 39) SASFLYS Light chain CDR3: (SEQ ID NO: 40) QQHYTTPPTVariable region: (SEQ ID NO: 41)DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ GTKVEIKRTV

The amino acid sequences of each CDR and variable region of the heavychain of the wild-type humanized anti-HER2 antibody are as follows.

Heavy chain CDR1: (SEQ ID NO: 42) DTYIH Heavy chain CDR2:(SEQ ID NO: 43) RIYPTNGYTRYADSVKG Heavy chain CDR3: (SEQ ID NO: 44)WGGDGFYAMDY Variable region: (SEQ ID NO: 45)EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG GDGFYAMDYWGQGTLVTVSS

The framework region (FR) is a region other than the CDRs present ineach variable region of the light chain and heavy chain of the antibody.FR plays a role of a scaffold linking the three CDRs and contributes tostructural stability of the CDR. Therefore, the amino acid sequence ofFR is highly conserved between antibodies of the same species. Eachvariable region of the light chain and heavy chain has three CDRs, CDR1,CDR2 and CDR3, and four FRs, FR1, FR2, FR3 and FR4. These are arrangedin the order of FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 from theN-terminal side of the variable region. Hereinafter, when referring toFR of an antibody, unless otherwise specified, the terms “frameworkregion” and “FR” mean FR of a light chain.

In the method for improving affinity of the present embodiment, at least3 of the amino acid residues satisfying predetermined conditions in theamino acid sequence of FR of a light chain of an unmodified antibody arechanged to charged amino acid residues. The amino acid residuesatisfying predetermined conditions is an amino acid residue satisfyingboth following conditions (1) and (2).

(1) In the amino acid sequence of FR of a light chain of an unmodifiedantibody, present at positions where total value of amino acidfrequencies of arginine, serine, threonine, valine, aspartic acid andglutamic acid is 35% or more.

(2) In the amino acid sequence of FR of a light chain of an unmodifiedantibody, has a ratio of solvent-exposed surface area of 20% or more.

In the present specification, in an unmodified antibody, changing anamino acid residue satisfying the above conditions (1) and (2) to acharged amino acid residue is also referred to as “modify” or“modification”. Hereinafter, an antibody obtained by modifying anunmodified antibody by the method for improving affinity of the presentembodiment is also referred to as a “modified antibody”.

Since the CDR is involved in specificity of the antibody, it ispreferable that the amino acid sequence of CDR is not changed in themethod for improving affinity of the present embodiment. That is, theamino acid sequence of the CDR of the modified antibody is preferablythe same as the amino acid sequence of the CDR of the unmodifiedantibody.

In the present embodiment, the affinity of the modified antibody for anantigen may be evaluated by a kinetic parameter in an antigen-antibodyreaction or may be evaluated by an immunological measurement method suchas an ELISA method. Examples of the kinetic parameter includedissociation constant (Kd), binding rate constant (kon), anddissociation rate constant (koff). Among them, Kd is preferable. Thekinetic parameter in an antigen-antibody reaction can be obtained bysurface plasmon resonance (SPR) technology or the like. The value of Kdin the antigen-antibody reaction of the modified antibody is, forexample, about ½, about ⅕, about 1/10, about 1/20, about 1/50, about1/100 or about 1/1000, as compared to the unmodified antibody.

As used herein, the term “position” refers to a position of an aminoacid residue in an amino acid sequence. In the present embodiment, theposition in the amino acid sequence of FR is a position in the aminoacid sequence of FR defined by a method of numbering amino acid residuesof CDR (hereinafter, also referred to as “numbering method”). Thenumbering method is a method for defining boundary and length of CDR andis known in the art. When the amino acid residues of CDR are numbered bythe numbering method, the amino acid residues of FR are also numbered.In the present embodiment, the position in the amino acid sequence of FRis indicated by the number assigned by the numbering method. As usedherein, “Ln” (where n is a positive integer) represents the nth positionin a light chain amino acid sequence. For example, L1 means a firstposition of the light chain amino acid sequence, and L2 means a secondposition of the light chain amino acid sequence.

Examples of the numbering method include Kabat method (Kabat E A. etal., Sequences of Proteins of Immunological Interest., NIH publicationNo. 91-3242), Chothia method (Chothia C. and Lesk A M., CanonicalStructures for the Hypervariable Regions of Immunoglobulins., J MolBiol., vol. 196, p. 901-917, 1987), IMGT method (Lefranc M P. et al.,Developmental and Comparative Immunology 29 (2005) 185-203), Honerggermethod (Honegger A. et al., Yet Another Numbering Scheme forImmunoglobulin Variable Domains: An Automatic Modeling and AnalysisTool., J Mol Biol., vol. 309, p. 657-670, 2001), ABM method, Contactmethod, and the like. In the present embodiment, the FR of an antibodymay be defined by any numbering method, but is preferably defined by theKabat method. In the unmodified antibody, in the Kabat method, FR1 ofthe light chain is defined as a region consisting of 1st to 23rd aminoacid residues of the light chain, FR2 of the light chain is defined as aregion consisting of 35th to 49th amino acid residues of the lightchain, FR3 of the light chain is defined as a region consisting of 57thto 88th amino acid residues of the light chain, and FR4 of the lightchain is defined as a region consisting of 98th to 109th amino acidresidues of the light chain.

Amino acid frequency is also called amino acid appearance frequency, andrefers to a ratio indicating how much predetermined amino acid appearsat each position of a plurality of amino acid sequences when aligningthese amino acid sequences. The amino acid frequency itself is a knownindex. Amino acid sequence alignment means aligning a plurality of aminoacid sequences in a comparable manner. Amino acid sequence alignment canbe performed by, for example, a known multiple alignment program such asClustalW or TREBMAL. A method for calculating amino acid frequencyitself is known, and amino acid frequency can be calculated by the abovemultiple alignment program or the like. Amino acid sequence alignmentand amino acid frequency calculation can also be performed by abYsis.abYsis can be used not only as a database to provide antibody sequences,but also as software for analyzing and predicting antibody sequences,structures and functions. For example, when an amino acid appears in allof a plurality of amino acid sequences at a predetermined position ofthe plurality of aligned amino acid sequences, amino acid frequency ofthe amino acid at that position is 100%. When an amino acid appears inhalf of a plurality of amino acid sequences at a predetermined positionof the plurality of aligned amino acid sequences, amino acid frequencyof the amino acid at that position is 50%. When an amino acid neverappears at a predetermined position of a plurality of aligned amino acidsequences, amino acid frequency of the amino acid at that position is0%.

In the present embodiment, amino acid sequences of light chains of aplurality of reference antibodies are obtained in order to specify aminoacid residues satisfying the above condition (1) in the FR of anunmodified antibody. By aligning the obtained amino acid sequences oflight chains of a plurality of reference antibodies, amino acidfrequencies at each position in an amino acid sequence of FRs of lightchains of reference antibodies can be obtained. Amino acid frequenciesat each position obtained from the plurality of reference antibodies canbe used as amino acid frequencies at corresponding positions in theamino acid sequence of a light chain of an unmodified antibody. Thereference antibody is not particularly limited as long as it is anantibody having an amino acid sequence different from that of theunmodified antibody. The reference antibody is preferably the same typeof antibody as the unmodified antibody, since the amino acid sequence ofFR of the antibody is highly conserved among antibodies of the sametype. The amino acid sequence of a light chain of a reference antibodycan be obtained from a known database such as GenBank, abYsis, and IMGT.The number of the amino acid sequence of a light chain of a referenceantibody is not particularly limited, but is, for example, 1000 or more,and preferably 10000 or more. The amino acid sequences of light chainsof a plurality of reference antibodies to be obtained may be amino acidsequences of the entire light chains or amino acid sequences of a partof the light chains. The amino acid sequence of a part of the lightchain preferably contains an amino acid sequence of FR1 and/or FR3.

By aligning the obtained amino acid sequences of light chains of aplurality of reference antibodies, respective amino acid frequencies ofarginine (R), serine (S), threonine (T), valine (V), aspartic acid (D)and glutamic acid (E) at each position of the amino acid sequences ofFRs of the reference antibodies are obtained. Here, the phrase “aligningthe amino acid sequences of light chains of a plurality of referenceantibodies” refers to aligning the amino acid sequences of light chainsof a plurality of reference antibodies so that numbers of the amino acidresidues in FR assigned by a predetermined numbering method match amongthe amino acid sequences of light chains of a plurality of referenceantibodies. In the present embodiment, it is preferable to align theamino acid sequences of light chains of a plurality of referenceantibodies so that the numbers of the amino acid residues in FR assignedby the Kabat method match. For each position, values of amino acidfrequencies of R, S, T, V, D and E are summed to calculate total value.For example, in the aligned amino acid sequences of light chains of aplurality of reference antibodies, total value X (%) of amino acidfrequencies at one position can be calculated from the numbers of R, S,T, V, D and E appearing at the position and the number of the obtainedamino acid sequences of light chains of reference antibodies, byfollowing formula (I). This calculation is performed for each positionof the aligned amino acid sequences containing FR1 and/or FR3 of lightchains of a plurality of reference antibodies. The number of the aminoacid sequences of light chains of reference antibodies used in thecalculation may differ from position to position. For example, the valueof X in L1 may be calculated based on 10000 amino acid sequences oflight chains of reference antibodies, and the value of X in L2 may becalculated based on 15000 amino acid sequences of light chains ofreference antibodies.

$\begin{matrix}{\left\lbrack {{Expression}\mspace{14mu} 1} \right\rbrack\mspace{596mu}} & \; \\{x = {\frac{\begin{matrix}{\left( {{Number}\mspace{14mu}{of}\mspace{14mu} R} \right) + \left( {{Number}\mspace{14mu}{of}\mspace{14mu} S} \right) + \left( {{Number}\mspace{14mu}{of}\mspace{14mu} T} \right) +} \\{\left( {{Number}\mspace{14mu}{of}\mspace{14mu} V} \right) + \left( {{Number}\mspace{14mu}{of}\mspace{14mu} D} \right) + \left( {{Number}\mspace{14mu}{of}\mspace{14mu} E} \right)}\end{matrix}}{\begin{matrix}\left( {{Number}\mspace{14mu}{of}\mspace{14mu}{amino}\mspace{14mu}{acid}\mspace{14mu}{sequence}\mspace{14mu}{of}\mspace{14mu}{light}\mspace{14mu}{chain}\mspace{14mu}{of}} \right. \\\left. {{reference}\mspace{14mu}{antibody}} \right)\end{matrix}} \times 100}} & (1)\end{matrix}$

Positions where the obtained total value of amino acid frequencies is35% or more and preferably 37% or more are specified in the alignedamino acid sequences of light chains of a plurality of referenceantibodies. Then, as the amino acid residues satisfying the abovecondition (1), amino acid residues present at positions corresponding tothe positions specified from the plurality of reference antibodies, inFR of a light chain of an unmodified antibody are specified. The aminoacid residues may be specified, for example, by aligning the amino acidsequence of a light chain of an unmodified antibody with the amino acidsequences of light chains of a plurality of reference antibodies. Whenthe amino acid sequences of light chains of a plurality of referenceantibodies are aligned, for example, based on FR defined by the Kabatmethod, amino acid residues in which the numbers of the amino acidresidues assigned by the Kabat method are the same as positions in thesequence of the reference antibody in which the total value of aminoacid frequencies is 35% or more in the FR of an unmodified antibody arespecified.

In the present embodiment, the amino acid residues satisfying the abovecondition (2) is specified based on the amino acid sequence of a lightchain of an unmodified antibody. Specifically, first, three-dimensionalstructure data of a light chain of an unmodified antibody is obtained byusing the amino acid sequence of a light chain of an unmodifiedantibody. The three-dimensional structure data includes coordinate dataof each amino acid residue constituting the protein, and data capable ofvisualizing a three-dimensional structure of the protein by a knownmolecular graphics program such as RasMol or Jmol is preferable. Thethree-dimensional structure data may be obtained by performing knownthree-dimensional structure analysis such as X-ray crystal structureanalysis and NMR analysis on the light chain of an unmodified antibody.In a preferred embodiment, the three-dimensional structure data isobtained by retrieving the amino acid sequence of a light chain of anunmodified antibody from a known protein three-dimensional structuredatabase such as Protein Data Bank (PDB) or Biological MagneticResonance Bank (BMRB). When the database has a protein having a sequencematching the amino acid sequence of a light chain of an unmodifiedantibody, three-dimensional structure data of the protein is downloaded.

When there is no three-dimensional structure data of a light chain of anunmodified antibody in the protein three-dimensional structure database,information necessary for creating the three-dimensional structure datamay be acquired, and three-dimensional structure data may be createdbased on the information. For example, three-dimensional structure datamay be created by a homology modeling method or the like, based on theamino acid sequence of a light chain of an unmodified antibody. In thehomology modeling method, as the information necessary for creatingthree-dimensional structure data, an amino acid sequence of a lightchain of an antibody having at least 20% identity with the amino acidsequence of a light chain of an unmodified antibody and having a knownthree-dimensional structure (hereinafter, also referred to as “referencesequence”) is used. In the art, it is known that proteins having highamino acid sequence identity are similar in three-dimensional structureto each other. The reference sequence can be obtained from a knowndatabase such as GenBank, abYsis, and IMGT. In the homology modelingmethod, the amino acid sequence of a light chain of an unmodifiedantibody and the reference sequence are aligned, and based on the resultof the alignment, three-dimensional structure of a light chain of anunmodified antibody is constructed from a known structure of thereference sequence. The creation of three-dimensional structure data bythe homology modeling method itself is known, and can be performed by aknown three-dimensional structure prediction program such as MODELLER.

Next, based on the obtained three-dimensional structure data, a ratio ofsolvent-exposed surface area of each amino acid residue of FR of anunmodified antibody is obtained. In the art, the solvent-exposed surfacearea is defined as a locus surface of a center of a probe sphere (1.4 Å)when the probe sphere assuming a water molecule is rolled so as to be incontact with a surface (Van der Waals surface) of a protein molecule.The solvent-exposed surface area itself is a known index. Thesolvent-exposed surface area of a protein can be obtained fromthree-dimensional structure data of the protein by a known program orsoftware such as SURFace, GETAREA, or Discovery Studio. It is alsopossible to obtain the solvent-exposed surface area of each amino acidresidue in the protein. The solvent-exposed surface area of the aminoacid residue in the protein depends on a size of side chain of the aminoacid. Therefore, the ratio of solvent-exposed surface area is used as anindex standardizing the solvent-exposed surface area of the amino acidresidue in the protein by the size of side chain of the amino acid. Theratio of solvent-exposed surface area itself is a known index. Forexample, ratio of solvent-exposed surface area Y (%) of amino acid X inprotein A is calculated by following formula (II). In the formula,“Ala-X-Ala” is a tripeptide consisting of a sequence in which the aminoacid X is sandwiched between two alanines.

$\begin{matrix}{\left\lbrack {{Expression}\mspace{14mu} 2} \right\rbrack\mspace{596mu}} & \; \\{Y = \frac{\begin{matrix}\left( {{Solvent}\text{-}{exposed}\mspace{14mu}{surface}\mspace{14mu}{area}\mspace{14mu}{of}\mspace{14mu}{amino}\mspace{14mu}{acid}\mspace{14mu} X} \right. \\\left. {{in}\mspace{14mu}{protein}\mspace{14mu} A} \right)\end{matrix}}{\begin{matrix}\left( {{Solvent}\text{-}{exposed}\mspace{14mu}{surface}\mspace{14mu}{area}\mspace{14mu}{of}\mspace{14mu}{amino}\mspace{14mu}{acid}\mspace{14mu} X} \right. \\\left. {{in}\mspace{14mu}{Ala}\text{-}X\text{-}{Ala}} \right)\end{matrix}}} & (2)\end{matrix}$

Then, in the amino acid sequence of FR of an unmodified antibody, aminoacid residues having an obtained ratio of solvent-exposed surface areaof 20% or more and preferably 25% or more are specified. As a result, inthe amino acid sequence of FR of an unmodified antibody, amino acidresidues present at positions where total value of amino acidfrequencies of R, S, T, V, D and E is 35% or more and having a ratio ofsolvent-exposed surface area of 20% or more can be specified.

Among the amino acid residues satisfying the above conditions (1) and(2), the number of amino acid residues to be changed to charged aminoacid residues is, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17 or 18. The charged amino acid residues may be basic aminoacid residues or acidic amino acid residues. Preferably, the chargedamino acid residues are amino acid residues of the same charge. Thecharged amino acid residues may be the same residues or differentresidues. The basic amino acid residue refers to a lysine residue, anarginine residue, and a histidine residue. The acidic amino acid residuerefers to an aspartic acid residue and a glutamic acid residue. In thepresent embodiment, as the charged amino acid residue, a basic aminoacid residue is preferable, and a lysine residue and an arginine residueare particularly preferable.

Among the amino acid residues satisfying the above conditions (1) and(2), the amino acid residues to be changed to charged amino acidresidues are preferably neutral amino acid residues. The neutral aminoacid residue refers to an alanine residue, an asparagine residue, anisoleucine residue, a glycine residue, a glutamine residue, a cysteineresidue, a threonine residue, a serine residue, a tyrosine residue, aphenylalanine residue, a proline residue, a valine residue, a methionineresidue, a leucine residue, and a tryptophan residue.

When the amino acid residue satisfying the above conditions (1) and (2)is a charged amino acid residue, the amino acid residue may be left asit is. Alternatively, when the amino acid residue satisfying the aboveconditions (1) and (2) is an acidic amino acid residue, the amino acidresidue may be changed to a basic amino acid residue. When the aminoacid residue satisfying the above conditions (1) and (2) is a basicamino acid residue, the amino acid residue may be changed to an acidicamino acid residue.

In the present embodiment, when electrical characteristic of CDR basedon the amino acid sequence of the CDR of the unmodified antibody isneutral, the amino acid residues satisfying the above conditions (1) and(2) may be changed to charged amino acid residues. The charged aminoacid residues are preferably basic amino acid residues. Here, theelectrical characteristic of CDR is an index uniquely defined by thepresent inventors. The electrical characteristic of CDR is determinedbased on the number of charged amino acid residues in the amino acidsequence of the CDR. Specifically, the electrical characteristic of CDRis determined by following formula (III).

Z=[Number of basic amino acid residues in amino acid sequence ofCDR]−[Number of acidic amino acid residues in amino acid sequence ofCDR]  (III)

wherein when Z is −1, 0 or 1, the electrical characteristic of CDR isneutral,

when Z is 2 or more, the electrical characteristic of CDR is positivelycharged, and

when Z is −2 or less, the electrical characteristic of CDR is negativelycharged.

The electrical characteristic of CDR may be determined based on theamino acid sequence of CDR of a light chain and/or a heavy chain. Whendetermining the electrical characteristic of CDR of the light chain, theamino acid sequence of the CDR in the formula (III) refers to all aminoacid sequences of CDR1, CDR2 and CDR3 of the light chain. Whendetermining the electrical characteristic of CDR of the heavy chain, theamino acid sequence of the CDR in the formula (III) refers to all aminoacid sequences of CDR1, CDR2 and CDR3 of the heavy chain. Preferably, itis determined based on the amino acid sequences of the CDRs of both thelight chain and heavy chain. In this case, the amino acid sequence ofthe CDR in the formula (III) refers to all amino acid sequences of CDR1,CDR2 and CDR3 of the light chain and CDR1, CDR2 and CDR3 of the heavychain. In the present embodiment, in an unmodified antibody whoseelectrical characteristic determined based on the amino acid sequencesof the CDRs of both the light chain and the heavy chain is neutral, atleast 3 of the amino acid residues satisfying the above conditions (1)and (2) may be changed to charged amino acid residues, and preferablybasic amino acid residues.

The amino acid sequence of the CDR can be obtained from a publicdatabase that discloses the sequence of the antibody gene.Alternatively, when there is a hybridoma that produces an unmodifiedantibody, the amino acid sequence of the CDR can be obtained byobtaining a nucleic acid encoding a heavy chain and a light chain fromthe hybridoma by a known method, and sequencing the base sequence of thenucleic acid.

The electrical characteristic of CDR differs depending on the antibody.For example, in the Kabat method, CDR of a light chain of a wild-type(i.e., unmodified) anti-lysozyme antibody used in the examples describedlater has one basic amino acid residue (arginine) and has no acidicamino acid residue. CDR of a heavy chain has one basic amino acidresidue (lysine) and three acidic amino acid residues (aspartic acid).Thus, the electrical characteristic of CDR of the wild-typeanti-lysozyme antibody is defined as neutral (Z=−1). The electricalcharacteristic of CDR of the anti-lysozyme antibody based on Chothiamethod is negatively charged.

The amino acid residue satisfying the above conditions (1) and (2) ispreferably an amino acid residue present in FR1 and/or FR3. Examples ofthe amino acid residue satisfying the above conditions (1) and (2) inFR1 and FR3 of the light chain include 1st, 3rd, 5th, 7th, 9th, 10th,12th, 14th, 17th, 18th, 20th, 22nd, 60th, 63rd, 65th, 67th, 69th, 70th,72nd, 74th, 76th, 77th, 79th and 81st amino acid residues of the lightchain defined by the Kabat method. Among them, the 1st, 3rd, 5th, 7th,9th, 10th, 12th, 14th, 17th, 18th, 20th and 22nd amino acid residues arepresent in FR1 of the light chain defined by the Kabat method, and the60th, 63rd, 65th, 67th, 69th, 70th, 72nd, 74th, 76th, 77th, 79th and81st amino acid residues are present in FR3 of the light chain definedby the Kabat method.

In a preferred embodiment, the amino acid residue satisfying the aboveconditions (1) and (2) are the 3rd, 5th, 9th, 17th, 18th, 20th, 22nd,60th, 63rd, 65th, 67th, 70th, 72nd, 74th, 76th, 77th, 79th and 81stamino acid residues of the light chain defined by the Kabat method.Among these amino acid residues, at least 3 amino acid residues to bechanged to charged amino acid residues preferably contain at least oneselected from the 3rd, 5th, 9th, 17th, 18th, 20th, 22nd, 60th, 74th,76th, 77th, 79th and 81st amino acid residues of the light chain definedby the Kabat method. More preferably, at least 3 amino acid residues tobe changed to charged amino acid residues preferably contain at least 3selected from the 3rd, 5th, 9th, 17th, 18th, 20th, 22nd, 60th, 74th,76th, 77th, 79th and 81st amino acid residues of the light chain definedby the Kabat method.

When modifying amino acid residues of FR1 of a light chain of anunmodified antibody, at least 3 amino acid residues to be changed tocharged amino acid residues are preferably selected from the 3rd, 5th,9th, 17th, 18th, 20th and 22nd amino acid residues of the light chaindefined by the Kabat method. Examples of the 3 amino acid residuesinclude any combination of following 1) to 4).

1) 3rd, 5th and 9th Amino acid residues of the light chain defined bythe Kabat method;

2) 17th, 18th and 20th Amino acid residues of the light chain defined bythe Kabat method;

3) 18th, 20th and 22nd Amino acid residues of the light chain defined bythe Kabat method; and

4) 5th, 9th and 22nd Amino acid residues of the light chain defined bythe Kabat method.

When modifying amino acid residues of FR3 of a light chain of anunmodified antibody, at least 3 amino acid residues to be changed tocharged amino acid residues are preferably selected from the 60th, 63rd,65th, 67th, 70th, 72nd, 74th, 76th, 77th, 79th and 81st amino acidresidues of the light chain defined by the Kabat method. Among theseamino acid residues, at least 3 amino acid residues to be changed tocharged amino acid residues preferably contain at least one selectedfrom 60th, 74th, 76th, 77th, 79th and 81st amino acid residues of thelight chain defined by the Kabat method. More preferably, at least 3amino acid residues to be changed to charged amino acid residuespreferably contain at least 3 selected from the 60th, 74th, 76th, 77th,79th and 81st amino acid residues of the light chain defined by theKabat method. Examples of the 3 amino acid residues include anycombination of following 5) to 8).

5) 60th, 76th and 77th Amino acid residues of the light chain defined bythe Kabat method;

6) 74th, 76th and 77th Amino acid residues of the light chain defined bythe Kabat method;

7) 77th, 79th and 81st Amino acid residues of the light chain defined bythe Kabat method; and

8) 76th, 77th and 81st Amino acid residues of the light chain defined bythe Kabat method.

Examples of the method of changing the amino acid residue satisfying theabove conditions (1) and (2) to a charged amino acid residue includesubstitution, insertion and the like of the amino acid residue. In themodification by inserting an amino acid residue, a charged amino acidresidue is inserted between the amino acid residue satisfying the aboveconditions (1) and (2) and an amino acid residue adjacent to theN-terminal side of the amino acid residue. For example, by inserting acharged amino acid residue between L2 amino acid residue and L3 aminoacid residue of the unmodified antibody, the L3 amino acid residue canbe changed to a charged amino acid residue. In a preferred embodiment,the amino acid residue satisfying the above conditions (1) and (2) issubstituted with a charged amino acid residue.

In the present embodiment, the amino acid residue of FR of an unmodifiedantibody can be changed to a charged amino acid residue by known methodssuch as DNA recombination technology and other molecular biologicaltechniques. For example, when there is a hybridoma that produces anunmodified antibody, RNA extracted from the hybridoma is used tosynthesize a polynucleotide encoding the light chain, by a reversetranscription reaction and a rapid amplification of cDNA ends (RACE)method. This polynucleotide is amplified by PCR using a primer formodifying at least 3 amino acid residues of FR to obtain apolynucleotide encoding a light chain in which FR has been modified. Theobtained polynucleotide is incorporated into an expression vector knownin the art to obtain an expression vector containing a polynucleotideencoding a modified antibody. The type of the expression vector is notparticularly limited, and it may be an expression vector for mammaliancells or an expression vector for E. coli. By transforming ortransfecting the obtained expression vector into an appropriate hostcell (for example, mammalian cell or E. coli), an antibody with improvedaffinity can be obtained.

When obtaining a modified antibody which is a single chain antibody(scFv), as shown in, for example, PCT International ApplicationPublication No. 2013/084371 A1, RNA extracted from the hybridoma may beused to synthesize a polynucleotide encoding a light chain variableregion by a reverse transcription reaction and PCR. This polynucleotideis ligated by overlap extension PCR or the like to obtain apolynucleotide encoding an unmodified scFv. The obtained polynucleotideis amplified by PCR using a primer for modifying at least 3 amino acidresidues of FR to obtain a polynucleotide encoding scFv in which FR hasbeen modified. The obtained polynucleotide is incorporated into anexpression vector known in the art to obtain an expression vectorcontaining a polynucleotide encoding a modified antibody in the form ofscFv. By transforming or transfecting the obtained expression vectorinto an appropriate host cell, a modified antibody in the form of scFvcan be obtained.

When there is no hybridoma that produces an unmodified antibody, anantibody-producing hybridoma may be prepared by known methods such asthose described in, for example, Kohler and Milstein, Nature, vol. 256,p. 495-497, 1975. Alternatively, RNA obtained from the spleen of ananimal such as a mouse immunized with an antigen of interest may beused. When using the RNA obtained from the spleen, for example, as shownin Fukunaga A and Tsumoto K, Protein Eng. Des. Sel. 2013, vol. 26, pp.773-780, a polynucleotide encoding an unmodified scFv having a desiredaffinity may be selected by a phage display method or the like, from theobtained polynucleotides encoding an unmodified scFv.

[2. Antibody with Improved Affinity and Production Method Thereof]

An antibody with improved affinity for an antigen as compared to anunmodified antibody of the present embodiment (hereinafter, alsoreferred to as “antibody with improved affinity”) is characterized inthat, in an amino acid sequence of FR of a light chain of an unmodifiedantibody, at least 3 of amino acid residues present at positions wheretotal value of amino acid frequencies of R, S, T, V, D and E is 35% ormore and having a ratio of solvent-exposed surface area of 20% or moreare changed to charged amino acid residues. The antibody with improvedaffinity is the same as the “modified antibody” described in thedescription of the method for improving affinity of the presentembodiment.

The antibody with improved affinity of the present embodiment ispreferably an antibody in which the amino acid residues of FR1 and/orFR3 of a light chain of an unmodified antibody are changed to chargedamino acid residues. Examples of the antibodies include antibodies inwhich at least 3 of the 1st, 3rd, 5th, 7th, 9th, 10th, 12th, 14th, 17th,18th, 20th, 22nd, 60th, 63rd, 65th, 67th, 69th, 70th, 72nd, 74th, 76th,77th, 79th and 81st amino acid residues of the light chain defined bythe Kabat method in the unmodified antibody are changed to charged aminoacid residues. In a preferred embodiment, the antibody with improvedaffinity is an antibody in which at least 3 amino acid residues selectedfrom the 3rd, 5th, 9th, 17th, 18th, 20th, 22nd, 60th, 63rd, 65th, 67th,70th, 72nd, 74th, 76th, 77th, 79th and 81st amino acid residues of thelight chain defined by the Kabat method in the unmodified antibody arechanged to charged amino acid residues. In this antibody, the above 3amino acid residues more preferably contain at least one selected fromthe 3rd, 5th, 9th, 17th, 18th, 20th, 22nd, 60th, 74th, 76th, 77th, 79thand 81st amino acid residues of the light chain defined by the Kabatmethod. The charged amino acid residues are preferably basic amino acidresidues.

When the antibody with improved affinity of the present embodiment is anantibody in which the amino acid residue of FR1 of a light chain of anunmodified antibody is modified, an antibody in which at least 3 of the3rd, 5th, 9th, 17th, 18th, 20th and 22nd amino acid residues of thelight chain defined by the Kabat method in the unmodified antibody arechanged to charged amino acid residues is preferable. Examples of the atleast 3 amino acid residues include any combination of the above 1) to4).

When the antibody with improved affinity of the present embodiment is anantibody in which the amino acid residue of FR1 of a light chain of anunmodified antibody is modified, an antibody in which at least 3 of the60th, 63rd, 65th, 67th, 70th, 72nd, 74th, 76th, 77th, 79th and 81stamino acid residues of the light chain defined by the Kabat method inthe unmodified antibody are changed to charged amino acid residues ispreferable. In particular, an antibody in which at least 3 selected fromthe 60th, 74th, 76th, 77th, 79th and 81st amino acid residues of thelight chain defined by the Kabat method are changed to charged aminoacid residues is preferable. Examples of the at least 3 amino acidresidues include any combination of the above 5) to 8).

The antibody with improved affinity of the present embodiment can beobtained by a method for producing an antibody with improved affinityfor an antigen as compared to an unmodified antibody of the presentembodiment (hereinafter, also referred to as “production method”). Inthe production method of the present embodiment, first, in the aminoacid sequence of FR of a light chain of an antibody (unmodifiedantibody), at least 3 of amino acid residues present at positions wheretotal value of amino acid frequencies of R, S, T, V, D and E is 35% ormore and having a ratio of solvent-exposed surface area of 20% or moreare changed to charged amino acid residues. Modifying the amino acidresidue in the unmodified antibody is the same as that described for themethod for improving affinity of the present embodiment.

Subsequently, the antibody obtained by the above modification isrecovered. For example, a host cell expressing an antibody with improvedaffinity is dissolved in a solution containing an appropriatesolubilizer to liberate the antibody in the solution. When the abovehost cell secretes an antibody into the medium, the culture supernatantis recovered. The liberated antibody can be recovered by methods knownin the art such as affinity chromatography. For example, when theproduced antibody is IgG, the antibody can be recovered by affinitychromatography using protein A or G. If necessary, the recoveredantibody may be purified by methods known in the art such as gelfiltration.

[3. Method for Analyzing Amino Acid Sequence of Antibody]

The scope of the present disclosure also includes a method for analyzingan amino acid sequence of an antibody (hereinafter, also referred to as“analysis method”). In the analysis method of the present embodiment,total value of amino acid frequencies of R, S, T, V, D and E at eachposition in the amino acid sequence of FR of a light chain of anantibody and a ratio of solvent-exposed surface area of each amino acidresidue in the amino acid sequence are obtained, and the obtained totalvalue of amino acid frequencies at each position and ratio ofsolvent-exposed surface area of each amino acid residue are output. Theoutput information is useful for examining candidates for amino acidresidues to be modified, for example, when modifying the analyzedantibody. Details of the total value of amino acid frequencies and theratio of solvent-exposed surface area are the same as those describedfor the method for improving affinity of the present embodiment. Theanalysis method of the present embodiment may be performed using, forexample, an amino acid sequence analysis system as shown in FIG. 1.

(Overview of Amino Acid Sequence Analysis System)

An example of the amino acid sequence analysis system will be describedbelow with reference to the drawings. However, the present embodiment isnot limited only to the embodiment shown in this example. With referenceto FIG. 1, the amino acid sequence analysis system includes an analysisdevice 10 of amino acid sequence and a sequence data server 20. Theanalysis device 10 and the sequence data server 20 are connected to eachother via a network 30 such as an intranet or an internet. In FIG. 1,the analysis device 10 is shown as a general-purpose computer systemincluding a control unit 100 (computer main body), an input unit 101,and a display unit 102, but the analysis device 10 is not limited tothis form.

When sequence information such as amino acid sequences of light chainsof a plurality of reference antibodies and three-dimensional structuredata of a light chain of an antibody is requested from the analysisdevice 10 to the sequence data server 20 via the network 30, thesequence information is downloaded from the sequence data server 20 tothe analysis device 10. The sequence data server 20 includes a databasein which information on amino acid sequences such as information on theamino acid sequences of light chains of a plurality of referenceantibodies and the three-dimensional structure data of a light chain ofan antibody are stored (hereinafter, also referred to as “amino acidsequence database”). The sequence data server 20 may be an external dataserver managed by a third party different from a user who uses theanalysis device 10. Examples of the database that can be used from theexternal data server (hereinafter, also referred to as “externaldatabase”) include GenBank, abYsis, IMGT, PDB, BMRB, and the like.

Although one sequence data server is shown in FIG. 1, the analysisdevice 10 may be connected to a plurality of sequence data servers. Theplurality of sequence data servers may include respectively differenttypes of amino acid sequence databases. For example, one sequence dataserver may include a database of amino acid sequence information for aprotein, including the amino acid sequences of light chains of aplurality of reference antibodies, and another sequence data server mayinclude a database of three-dimensional structure data of the proteinincluding the light chain of an antibody.

The analysis device 10 obtains an amino acid sequence of a light chainof an unmodified antibody from an input by the user or a storage medium40 described later. The analysis device 10 requests the sequence dataserver 20 via the network 30 for the amino acid sequences of lightchains of a plurality of reference antibodies and the three-dimensionalstructure data of a light chain of an antibody. The sequence data server20 provides the requested data to the analysis device 10 via the network30. Then, the analysis device 10 analyzes the amino acid sequence of theunmodified antibody using the obtained sequence.

(Hardware Configuration of Amino Acid Sequence Analysis System)

With reference to FIG. 2, the control unit 100 of the analysis device 10includes Central Processing Unit (CPU) 110, memory 111, a storage device112, an interface 113, a reading device 114, and a bus 115 fordata-communicably connecting them. The control unit 100 is communicablyconnected to external devices such as the input unit 101 and the outputunit 102 and the network 30 via the interface 113. The storage medium 40is a computer-readable, non-transitory tangible storage medium such as aCD-ROM or a USB memory. The sequence data server 20 includes a controlunit 210, an input unit 211, an output unit 212, a storage unit 213, anda communication unit 214. Amino acid sequence database 215 is stored inthe storage unit 213. The sequence data server 20 is communicablyconnected to the network 30 via the communication unit 214. The inputunits 101 and 211 are input devices such as a mouse and a keyboard. Theoutput units 102 and 212 are display devices such as a liquid crystaldisplay.

The CPU 110 executes a computer program of the present embodiment storedin the memory 111 or the storage device 112 to perform a processdescribed later. The memory 111 includes Read Only Memory (ROM) andRandom Access Memory (RAM). The ROM includes, for example, mask ROM,PROM, EPROM, EEPROM, and the like. In the ROM, a computer programexecuted by the CPU 110 and data used for executing the computer programare recorded. The RAM includes, for example, SRAM, DRAM, and the like.The RAM is used for reading the program recorded in the ROM and thestorage device 112. The RAM is used as a work area of the CPU 110 whenthese programs are executed.

The storage device 112 includes, for example, a hard disk. In thestorage device 112, programs such as operating systems and applicationprograms for execution by the CPU 110, and data used for executing theprograms are stored. Examples of the application program include thecomputer program of the present embodiment, a program for executingamino acid sequence alignment, a program for executing amino acidfrequency calculation, a program for executing calculation of ratio ofsolvent-exposed surface area, and the like. Further, in the storagedevice 112, respective threshold values of the amino acid frequency andthe ratio of solvent-exposed surface area may be recorded.

The reading device 114 includes a CD-ROM drive, a DVD-ROM drive, a USBport, an SD card reader, a CF card reader, a memory stick reader, asolid state drive, a flexible disk drive, and the like. The readingdevice 114 can read data recorded on the storage medium 40 (e.g.,information on the amino acid sequences of light chains of an unmodifiedantibody and/or a plurality of reference antibodies and/or the nucleicacid sequence encoding them, and the like), and a computer program.

(Process Procedure of Analysis Device of Amino Acid Sequence)

With reference to FIG. 3, an analysis process of amino acid sequence ofan antibody executed by the analysis device 10 will be described. Here,using the amino acid sequences of light chains of a plurality ofreference antibodies and the three-dimensional structure data of a lightchain of an unmodified antibody downloaded from an external database, acase of outputting total value of amino acid frequencies at eachposition and a ratio of solvent-exposed surface area of each amino acidresidue will be described as an example. However, the present embodimentis not limited only to this example. Unless otherwise specified in thefollowing description, the process performed by the control unit 100means the process performed by the CPU 110.

In step S10, the control unit 100 downloads data of the amino acidsequences of light chains of a plurality of reference antibodies fromthe amino acid sequence database 215 of the sequence data server 20 viathe network 30, and the control unit 100 stores the data in the storagedevice 112. The amino acid sequence to be downloaded may be a sequenceof the entire light chain or a sequence of a part of the light chaincontaining an amino acid sequence of FR1 and/or FR3. When the useralready has the data of the amino acid sequences of light chains of aplurality of reference antibodies, the control unit 100 may obtain thedata of the amino acid sequences input by the user instead ofdownloading. The input may be performed by the input unit 101, or theinput may be performed by transferring the data of the amino acidsequences stored in the storage medium 40 to the storage device 112.

In step S11, the control unit 100 aligns the obtained amino acidsequences of light chains of a plurality of reference antibodies.Specifically, the control unit 100 aligns the amino acid sequences of aplurality of reference antibodies so that the numbers of the amino acidresidues in FR assigned by the Kabat method match. Alignment may beperformed based on a numbering method other than the Kabat method. Instep S12, the control unit 100 calculates total value of amino acidfrequencies of R, S, T, V, D and E at each position of the amino acidsequences of FRs of a plurality of reference antibodies. Thiscalculation is the same as that described for the method for improvingaffinity of the present embodiment.

In step S13, the control unit 100 obtains an amino acid sequence of alight chain of an unmodified antibody. The amino acid sequence of alight chain of an unmodified antibody to be obtained may be an aminoacid sequence of the entire light chain or an amino acid sequence of apart of the light chain. The amino acid sequence of a part of the lightchain preferably contains an amino acid sequence of FR1 and/or FR3, andmore preferably further contains an amino acid sequence of CDR. In apreferred embodiment, the control unit 100 obtains the amino acidsequence of the entire light chain of an unmodified antibody. The aminoacid sequence may be input by the user, or the amino acid sequence maybe previously stored in the storage medium 40 or the storage device 112.When the amino acid sequence of a light chain of an unmodified antibodyis present in the amino acid sequence database 215, the control unit 100may download the data of the amino acid sequence. In step S14, thecontrol unit 100 specifies amino acid residues present at positionscorresponding to the positions specified from the plurality of referenceantibodies in FR of an unmodified antibody are specified. The controlunit 100 may specify the amino acid residues by aligning the amino acidsequence of a light chain of an unmodified antibody with the amino acidsequences of light chains of a plurality of reference antibodies. As aresult, the control unit 100 obtains the calculated total value of aminoacid frequencies as total value of amino acid frequencies at eachposition of the amino acid sequence of FR of an unmodified antibody, andthe control unit 100 stores the total value in the storage device 112.

In step S15, the control unit 100 downloads three-dimensional structuredata of a light chain of an unmodified antibody from the amino acidsequence database 215, based on the amino acid sequence of a light chainof an unmodified antibody. The control unit 100 retrieves the amino acidsequence of a light chain of an unmodified antibody from the amino acidsequence database 215. When the database contains a protein having asequence matching the amino acid sequence of a light chain of anunmodified antibody, the control unit 100 downloads three-dimensionalstructure data of the protein, and the control unit 100 stores the datain the storage device 112 as the three-dimensional structure data of alight chain of an unmodified antibody.

In step S16, the control unit 100 calculates a ratio of solvent-exposedsurface area of each amino acid residue of FR of an unmodified antibody,based on the obtained three-dimensional structure data, and the controlunit 100 stores the ratio in the storage device 112. This calculation isthe same as that described for the method for improving affinity of thepresent embodiment. When the user has the three-dimensional structuredata of a light chain of an unmodified antibody, the control unit 100may obtain the three-dimensional structure data input by the userinstead of downloading. The input may be performed by the input unit101, or the input may be performed by transferring the three-dimensionalstructure data stored in the storage medium 40 to the storage device112. When there is no three-dimensional structure data of a light chainof an unmodified antibody in the amino acid sequence database 215, thecontrol unit 100 may acquire information necessary for creating thethree-dimensional structure data from the database, and the control unit100 may create three-dimensional structure data based on theinformation. Specifically, the control unit 100 retrieves for areference sequence from the amino acid sequence database 215, and thecontrol unit 100 downloads its amino acid sequence. Subsequently, thecontrol unit 100 aligns the reference sequence with the amino acidsequence of the unmodified antibody. Then, the control unit 100 createsthree-dimensional structure data of a light chain of an unmodifiedantibody from a known structure of the reference sequence based on thealignment result. The creation of the three-dimensional structure datais the same as that described for the method for improving affinity ofthe present embodiment.

In step S17, the control unit 100 transmits the total value of aminoacid frequencies at each position of the amino acid sequence of FR of anunmodified antibody specified in step S14, and the ratio ofsolvent-exposed surface area of each amino acid residue of FR of anunmodified antibody calculated in step S16 to the output unit 102. FIG.4 shows an example of a screen of analysis results displayed on theoutput unit 102. This screen shows the total value of amino acidfrequencies and the ratio of solvent-exposed surface area at eachposition (L1 to L23) of the amino acid sequence of FR1 of an unmodifiedantibody. However, the display of the screen is not limited to thisexample.

In the process shown in FIG. 3, step S13 may be performed before stepS10, S11 or S12. Steps S15 and S16 may be performed before step S10, S11or S12 as long as it is after the step of obtaining the amino acidsequence of the unmodified antibody (step S13). Steps S10 and S15 may beperformed at the same time as long as it is after the step of obtainingthe amino acid sequence of the unmodified antibody (step S13).

[4. Method for Specifying Candidates for Antibody Modification Sites]

The scope of the present disclosure also includes a method forspecifying candidates for antibody modification sites (hereinafter, alsoreferred to as a “specification method”). In the specification method ofthe present embodiment, total value of amino acid frequencies of R, S,T, V, D and E at each position in the amino acid sequence of FR of alight chain of an antibody and a ratio of solvent-exposed surface areaof each amino acid residue in the amino acid sequence are obtained, andan amino acid residue present at positions where total value of aminoacid frequencies is 35% or more and having a ratio of solvent-exposedsurface area of 20% or more are changed to charged amino acid residuesis specified as candidates for a site to be modified to improveaffinity. Details of the total value of amino acid frequencies and theratio of solvent-exposed surface area are the same as those describedfor the method for improving affinity of the present embodiment.

In the amino acid sequence of a light chain of an antibody, at least 3of the amino acid residues specified by the specification method of thepresent embodiment are changed to charged amino acid residues, so thataffinity of the antibody for an antigen can be improved as compared tothat of the unmodified antibody. Then, by recovering the antibody inwhich the amino acid residue is modified, a modified antibody withimproved affinity of the antibody for an antigen can be obtained. Themodification of amino acid residue and the recovery of antibody are thesame as those described for the method for improving affinity of thepresent embodiment and the production method of the present embodiment.

The specification method of the present embodiment can be performed byan amino acid sequence analysis system as shown in FIG. 1, similarly tothe analysis method of the present embodiment. Outline and hardwareconfiguration of the amino acid sequence analysis system for performingthe specification method of the present embodiment are the same as thosedescribed for the analysis method of the present embodiment.

(Process Procedure of Analysis Device of Amino Acid Sequence)

With reference to FIG. 5, an analysis process of amino acid sequence forspecifying candidates for antibody modification sites executed by theanalysis device 10 will be described. Here, using the amino acidsequences of light chains of a plurality of reference antibodies and thethree-dimensional structure data of a light chain of an unmodifiedantibody downloaded from an external database, a case of calculatingtotal value of amino acid frequencies at each position and a ratio ofsolvent-exposed surface area of each amino acid residue will bedescribed as an example. However, the present embodiment is not limitedonly to this example. Unless otherwise specified in the followingdescription, the process performed by the control unit 100 means theprocess performed by the CPU 110. Details of steps S20 to S26 are thesame as those described for steps S10 to S16.

In step S27, the control unit 100 specifies amino acid residues presentat positions where total value of amino acid frequencies of R, S, T, V,D and E, among positions of the amino acid sequence of FR of anunmodified antibody specified in step S24, is 35% or more, and having aratio of solvent-exposed surface area calculated in step S26 of 20% ormore. Then, in step S28, the control unit 100 transmits the amino acidresidues specified in step S27 to the output unit 102. FIG. 6 shows anexample of a screen of analysis results displayed on the output unit102. This screen shows the total value of amino acid frequencies and theratio of solvent-exposed surface area at each position (L1 to L23) ofthe amino acid sequence of FR1 of a light chain of an unmodifiedantibody. In this screen, the positions and values of the amino acidresidues specified in step S27 are highlighted in bold and markers.However, the display of the screen is not limited to this example. Thespecified amino acid residues are presented to the user as candidatesfor modification sites in the unmodified antibody.

With reference to FIG. 7, an analysis process of amino acid sequence forcreating a sequence when the candidates for antibody modification sitesare changed to charged amino acid residues executed by the analysisdevice 10 will be described. Here, using the amino acid sequences oflight chains of a plurality of reference antibodies and thethree-dimensional structure data of a light chain of an unmodifiedantibody downloaded from an external database, a case of calculatingtotal value of amino acid frequencies at each position and a ratio ofsolvent-exposed surface area of each amino acid residue will bedescribed as an example. However, the present embodiment is not limitedonly to this example. Unless otherwise specified in the followingdescription, the process performed by the control unit 100 means theprocess performed by the CPU 110. Steps S30 to S36 are the same asdescribed for steps S10 to S16, and step S37 is the same as step S27.

In step S38, the control unit 100 creates an amino acid sequence and/ora base sequence encoding the sequence when at least 3 of the amino acidresidues specified in step S37 are changed to charged amino acidresidues. When creating the amino acid sequence, the control unit 100creates an amino acid sequence in which at least 3 of the amino acidresidues specified in step S37 are changed to R, K, D or E (amino acidsequence of a light chain of a modified antibody), in the amino acidsequence of a light chain of an unmodified antibody obtained in stepS33, and the control unit 100 stores the amino acid sequence in thestorage device 112. Which amino acid residue is changed to the chargedamino acid residue may be preset or may be determined by the user. Whichof R, K, D and E is selected as the charged amino acid residue may bepreset or may be determined by the user.

When creating a base sequence encoding the amino acid sequence of alight chain of a modified antibody, the control unit 100 creates anamino acid sequence in which at least 3 of the amino acid residuesspecified in step S37 are changed to R, K, D or E, in the amino acidsequence obtained in step S33, the control unit 100 converts it into abase sequence, and the control unit 100 stores the base sequence in thestorage device 112. When there are a plurality of codons for one aminoacid, which codon is selected may be preset or may be determined by theuser. When creating a base sequence, the control unit 100 may obtain abase sequence encoding the light chain of an unmodified antibody in stepS33. When the nucleotide sequence is obtained, the control unit 100 maycreate a base sequence encoding the amino acid sequence of a light chainof a modified antibody, by changing codons corresponding to the aminoacid residues specified in step S37 to codons corresponding to thecharged amino acid residues. Then, in step S39, the control unit 100transmits the amino acid residues specified in step S37, the amino acidsequence of a light chain of a modified antibody and/or the basesequence encoding the amino acid sequence created in step S38 to theoutput unit 102.

In the specification method of the present embodiment, the electricalcharacteristic of CDR based on the amino acid sequence of the CDR of theunmodified antibody may be calculated and output. The electricalcharacteristic of CDR is the same as that described for the method forimproving affinity of the present embodiment. When the calculatedelectrical characteristic of CDR is neutral, a modified amino acidsequence when at least 3 of the amino acid residues specified ascandidates for modification sites are changed to charged amino acidresidues (preferably basic amino acid residues) may be created, and themodified amino acid sequence may be output.

With reference to FIG. 8, an analysis process of amino acid sequence forcreating a sequence when candidates for antibody modification sites arechanged to basic amino acid residues when the electrical characteristicof CDR is neutral executed by the analysis device 10 will be described.Here, using the amino acid sequences of light chains of a plurality ofreference antibodies and the three-dimensional structure data of a lightchain of an unmodified antibody downloaded from an external database, acase of calculating total value of amino acid frequencies at eachposition and a ratio of solvent-exposed surface area of each amino acidresidue will be described as an example. However, the present embodimentis not limited only to this example. Unless otherwise specified in thefollowing description, the process performed by the control unit 100means the process performed by the CPU 110. Steps S40 to S46 are thesame as described for steps S10 to S16, and step S47 is the same as stepS27.

In step S48, the control unit 100 calculates the electricalcharacteristic of CDR of the unmodified antibody, based on the aminoacid sequence of a light chain of an unmodified antibody obtained instep S43. The electrical characteristic of CDR is calculated by theabove formula (III). In step S49, whether or not the calculatedelectrical characteristic of CDR is neutral is determined. When thevalue calculated by the above formula (III) is −1, 0 or 1, theelectrical characteristic of CDR is determined to be neutral, and theprocess proceeds to step S50. In step S50, the control unit 100 createsan amino acid sequence and/or a base sequence encoding the sequence whenat least 3 of the amino acid residues specified in step S47 are changedto basic amino acid residues. Then, in step S51, the control unit 100transmits the amino acid residues specified in step S47, the electricalcharacteristic of CDR calculated in step S48, the amino acid sequence ofa light chain of a modified antibody created in step S50 and/or the basesequence encoding the amino acid sequence to the output unit 102. Instep S49, when the value calculated by the above formula (III) is −2 orless or 2 or more, the electrical characteristic of CDR is determinednot to be neutral, and the process proceeds to step S52. In step S52,the control unit 100 transmits the amino acid residues specified in stepS47 and the electrical characteristic of CDR calculated in step S48 tothe output unit 102.

Hereinafter, the present disclosure will be described in more detail byexamples, but the present disclosure is not limited to these examples.

EXAMPLES Example 1 Preparation of Antibody in which Amino Acid Residueof FR1 or FR3 of Light Chain is Modified

Variants of each antibody were prepared by substituting 3 amino acidresidues of FR1 or FR3 of an anti-lysozyme antibody with charged aminoacid residues.

(1) Obtainment of Gene of Wild-Type Anti-Lysozyme Antibody

Synthesis of anti-lysozyme antibody gene was entrusted to GenScriptJapan Inc. to obtain a plasmid DNA containing wild-type anti-lysozymeantibody gene.

(2) Preparation of Variant of Anti-Lysozyme Antibody [Reagents]

QIAprep Spin Miniprep kit (QIAGEN)

PrimeSTAR (registered trademark) Max DNA Polymerase (Takara Bio Inc.)

Ligation high ver.2 (TOYOBO CO., LTD.)

T4 Polynucleotide Kinase (TOYOBO CO., LTD.)

Dpn I (TOYOBO CO., LTD.)

Competent high DH5α (TOYOBO CO., LTD.)

(2.1) Primer Design and PCR

In order to substitute predetermined 3 amino acid residues of lightchain FR1 or FR3 of a wild-type anti-lysozyme antibody with arginineresidues, PCR was performed using the plasmid obtained in the above (1)and primers represented by following base sequences. Three numbers inname of each primer indicate positions of amino acid residuessubstituted with arginine residues in FR of the light chain defined bythe Kabat method. Primers of SEQ ID NOs: 1 to 11 were used as forwardprimers, and primers of SEQ ID NOs: 12 to 22 were used as reverseprimers.

[Primer for Preparing Variants]

2, 4, 6 Variant FOR: (SEQ ID NO: 1)5′ AGAACCAGAAGCCCGGCGACCCTCTCGGTCACCCCCGGC 3′ 2,4, 8 Variant FOR:(SEQ ID NO: 2) 5′ AGAGCAGAGCGACCCTCTCGGTCACCCCCGGC 3′3, 5, 9 Variant FOR: (SEQ ID NO: 3) 5′ GCCCGCGCACCCTCTCGGTCACCCCCGGC 3′4, 8, 13 Variant FOR: (SEQ ID NO: 4)5′ CCCTCTCGAGAACCCCCGGCAACTCGGTGTCGC 3′ 5, 9, 22 Variant FOR:(SEQ ID NO: 5) 5′ GGCAACTCGGTGTCGCTCCGCTGCCGCGCCTCGCAGTCG 3′13, 16, 19 Variant FOR: (SEQ ID NO: 6)5′ AACTCGCGATCGCTCTCGTGCCGCGCCTCGCAGTCG 3′ 16, 21, 23 Variant FOR:(SEQ ID NO: 7) 5′ GTGTCGCGATCGCGACGCGCCTCGCAGTCGATCGGC 3′17, 18, 20 Variant FOR: (SEQ ID NO: 8) 5′ CTCTCGTGCCGCGCCTCGCAG 3′18, 20, 22 Variant FOR: (SEQ ID NO: 9) 5′ CGCTGCCGCGCCTCGCAGTCGATCGGC 3′19, 21, 23 Variant FOR: (SEQ ID NO: 10)5′ AGATCGAGACGCGCCTCGCAGTCGATCGGC 3′ 63, 65, 67 Variant FOR:(SEQ ID NO: 11) 5′ GGCACCGACTTCACCCTGTCG 3′ 2, 4, 6 Variant REV:(SEQ ID NO: 12) 5′ GACTCTATCTCCTCTGGACATTATGACTGAGGC 3′2, 4, 8 Variant REV: (SEQ ID NO: 13)5′ GGGTTCTGACTCTATCTCCTCTGGACATTATGACTGAGGC 3′ 3, 5, 9 Variant REV:(SEQ ID NO: 14) 5′ TCTGGCGCAGGCGGATATCTCCTCTGGACATTATG 3′4, 8, 13 Variant REV: (SEQ ID NO: 15)5′ TCGCTCTGCTCTGGGTTCTGACGATATCTCCTCTGGACATTATG 3′ 5, 9, 22 Variant REV:(SEQ ID NO: 16) 5′ GGGGGTGACCGAGAGGGTGCGCGGGCTCTGGCGCAGGACGATATCTCCTCTGG 3′ 13, 16, 19 Variant REV: (SEQ ID NO: 17)5′ TCGGGGGGTTCGCGAGAGGGTCGCCGGGCTCTGGG 3′ 16, 21, 23 Variant REV:(SEQ ID NO: 18) 5′ CGAGTTTCGGGGGGTGACCGAGAGGGTCGC 3′17, 18, 20 Variant REV: (SEQ ID NO: 19)5′ GCGCACGCGGCGGCCGGGGGTGACCGAGAGGG 3′ 18, 20, 22 Variant REV:(SEQ ID NO: 20) 5′ GAGGCGCACGCGGTTGCCGGGGGTGACCGAGAGGG 3′19, 21, 23 Variant REV: (SEQ ID NO: 21)5′ CGATCTCGAGTTGCCGGGGGTGACCGAGAGGG 3′ 63, 65, 67 Variant REV:(SEQ ID NO: 22) 5′ TCTGCCTCTGCCTCTGAAGCGCGACGGGATCCCCG 3′

Using the plasmid obtained in the above (1) as a template, a PCRreaction solution with the following composition was prepared.

[PCR Reaction Solution]

PrimeSTAR (registered trademark) Max DNA Polymerase 12.5 μL Forwardprimer (10 μM) 1 μL Reverse primer (10 μM) 1 μL Template plasmid (3ng/μL) 1 μL Purified water 9.5 μL Total 25 μL

The prepared PCR reaction solution was subjected to a PCR reaction underthe following reaction conditions.

[Reaction Conditions]

30 cycles at 98° C. for 10 seconds, 98° C. for 10 seconds, 54° C. for 10seconds and 72° C. for 45 seconds, and at 72° C. for 3 minutes.

The obtained PCR product was fragmented by adding 1 μL of DpnI (10 U/μL)to the PCR product (25 μL). Using the DpnI-treated PCR product, aligation reaction solution with the following composition was prepared.The reaction solution was incubated at 16° C. for 1 hour to perform aligation reaction.

[Ligation Reaction Liquid]

DpnI-treated PCR product 2 μL Ligation high ver.2 5 μL T4 Polynucleotidekinase 1 μL Purified water 7 μL Total 15 μL

(2.2) Transformation, Plasmid Extraction and Sequence Confirmation

The solution (3 μL) after the ligation reaction was added to DH5α (30μL), and the mixture was allowed to stand on ice for 30 minutes.Thereafter, the mixture was heat shocked by heating at 42° C. for 45seconds. The mixture was again allowed to stand on ice for 2 minutes,then the whole amount was applied to an ampicillin-containing LB plate.The plate was incubated at 37° C. for 16 hours to obtain E. colitransformants. Single colonies on the plate were placed in theampicillin-containing LB liquid medium, and the medium wasshake-cultured (250 rpm) at 37° C. for 16 hours. Plasmids were extractedfrom the obtained E. coli using the QIAprep Spin Miniprep kit. The basesequence of each obtained plasmid was confirmed using pcDNA 3.4 vectorprimer. Hereinafter, these plasmids were used as plasmids for expressingmammalian cells.

(3) Expression in Mammalian Cells [Reagents]

Expi293 (trademark) Cells (Invitrogen)

Expi293 (trademark) Expression medium (Invitrogen)

ExpiFectamine (trademark) 293 transfection kit (Invitrogen)

(3.1) Transfection

Expi293 Cells were proliferated by shaking culture (150 rpm) at 37° C.in a 5% CO₂ atmosphere. 30 mL of cell culture (3.0×10⁶ cells/mL) wasprepared according to the number of samples. A DNA solution with thefollowing composition was prepared using a plasmid encoding each variantand a plasmid encoding a wild-type antibody. The DNA solution wasallowed to stand for 5 minutes.

[DNA Solution]

Light chain plasmid solution Amount (μL) corresponding to 15 μg Heavychain plasmid solution Amount (μL) corresponding to 15 μg Opti-MEM(trademark) Appropriate amount (mL) Total 1.5 mL

A transfection reagent with the following composition was prepared. Thetransfection reagent was allowed to stand for 5 minutes.

ExpiFectamine reagent 80 μL Light chain plasmid solution 1420 μL Total1.5 mL

The prepared DNA solution and the transfection reagent were mixed. Themixture was allowed to stand for 20 minutes. The resulting mixture (3mL) was added to the cell culture (30 mL). The mixture wasshake-cultured (150 rpm) at 37° C. for 20 hours in a 5% CO₂ atmosphere.After 20 hours, 150 μL and 1.5 mL of ExpiFectamine (trademark)transfection enhancers 1 and 2 were added to each culture, respectively.Each mixture was shake-cultured (150 rpm) at 37° C. for 6 days in a 5%CO₂ atmosphere.

(3.2) Recovery and Purification of Antibody

Each cell culture was centrifuged at 3000 rpm for 5 minutes, and theculture supernatant was recovered. The culture supernatant contains eachantibody secreted from transfected Expi293 (trademark) cells. Theobtained culture supernatant was again centrifuged at 15000×G for 10minutes, and the supernatant was recovered. To the obtained supernatant(30 mL) was added 100 μL of antibody purification carrier Ni SepharoseHigh Performance (GE Healthcare), and the mixture was reacted at roomtemperature for 2 hours. The carrier was recovered to remove thesupernatant, and TBS (1 mL) was added to wash the carrier. To thecarrier was added 1000 μL of TBS containing 100 mM imidazole to elutethe antibody captured on the carrier. This elution operation wasperformed a total of 3 times to obtain an antibody solution.

(4) Measurement of Affinity

The affinity of the prepared variants was measured using Biacore(registered trademark) T200 (GE Healthcare). Chicken egg white-derivedlysozyme (Sigma-Aldrich) was used as an antigen for the anti-lysozymeantibody. Antigen was immobilized (immobilization: 50 RU) to a sensorchip for Biacore (registered trademark) Series S Sensor Chip CMS (GEHealthcare). The antibody solution was diluted to prepare antibodysolutions of 30 nM, 15 nM, 7.5 nM, 3.75 nM and 1875 nM. The antibodysolutions at each concentration were delivered to Biacore (registeredtrademark) T200 (GE Healthcare) (association time of 120 seconds anddissociation time of 1800 seconds). Measurement data was analyzed usingBiacore (registered trademark) Evaluation software, and the data on theaffinity of each antibody was obtained. Kd values of each antibody areshown in Table 2.

TABLE 2 Anti-lysozyme antibody K_(d) (M) Wild-type 1.31E−10 63, 65, 67Variant 9.20E−11 18, 20, 22 Variant 6.52E−11 3, 5, 9 Variant 7.23E−1117, 18, 20 Variant 1.05E−10 5, 9, 22 Variant 6.90E−11

(5) Result

As shown in Table 2, Kd values of 63, 65, 67 variant in which FR3 of alight chain was modified, 18, 20, 22 variant in which FR1 of a lightchain was modified, 3, 5, 9 variant, 17, 18, 20 variant, 18, 20, 22variant and 5, 9, 22 variant were lower than Kd value of the wild-typeanti-lysozyme antibody. Therefore, the variants shown in Table 2 hadimproved affinity for an antigen as compared to the wild-type bychanging 3 amino acid residues of FR3 or FR1 to basic amino acidresidues.

On the other hand, for the variants not shown in Table 2, antibodyexpression was not observed in the first place. Expression was confirmedby analyzing the eluate obtained in the above (3.2) by SDS-PAGE and CBBstaining. The results are shown in FIG. 9. As can be seen from FIG. 9,heavy chain and light chain bands of the antibody were observed in lanes1 and 2. Therefore, expression of wild-type antibody and 63, 65, 67variant was observed. However, no antibody band was observed in lanes 3to 8, so that expression of 2, 4, 6 variant, 2, 4, 8 variant, 4, 8, 13variant, 19, 21, 23 variant, 13, 16, 19 variant and 16, 21, 23 variantwere not observed.

Example 2 Characteristics of Antibody with Improved Affinity for Antigen

In order to find features common to the variants of Example 1 withimproved affinity for an antigen, the present inventors calculated aminoacid frequencies at each position of FR1 and FR3 of the light chains.The present inventors considered that a side chain of the amino acidresidue facing the surface of the antibody molecule is involved in theimprovement in affinity for an antigen, and calculated ratios ofsolvent-exposed surface areas of each amino acid residue of FR1 and FR3of the light chains.

(1) Amino Acid Frequency

Amino acid sequences of light chains of about 30,000 mouse antibodieswere downloaded as reference antibodies from abYsis, a public databasethat provides amino acid sequences of antibodies. The obtained aminoacid sequences of light chains of reference antibodies were aligned sothat the numbers of the amino acid residues in FR of the light chainsassigned by Kabat method matched. Amino acid frequencies at eachposition of FR1 and FR3 of the obtained light chains of referenceantibodies were obtained. Sequence alignment and amino acid frequencieswere obtained by abYsis. It was found that appearance frequencies of R,S, T, V, D and E tended to be high at positions corresponding to theamino acid residues modified by the variants of Example 1 with improvedaffinity for an antigen. Therefore, total value of these amino acidfrequencies was calculated at each position of FR1 and FR3 of the lightchains. The total value of amino acid frequencies was calculated by theabove formula (I). Here, the obtained numbers by the Kabat methodassigned to FRs of light chains of reference antibodies were the samefor FR of a light chain of a wild-type anti-lysozyme antibody.Therefore, the total value of amino acid frequencies obtained from theamino acid sequences of light chains of reference antibodies was used asa value for the amino acid sequence of a light chain of a wild-typeanti-lysozyme antibody.

(2) Ratio of Solvent-Exposed Surface Area

An amino acid sequence of a light chain of a wild-type anti-lysozymeantibody was retrieved from PDB, a public database that providesthree-dimensional structure data of proteins, and three-dimensionalstructure data of a light chain of the antibody was downloaded. Usingthe obtained three-dimensional structure data, ratios of solvent-exposedsurface areas of each amino acid residue of FR1 and FR3 of a light chainof a wild-type anti-lysozyme antibody were obtained by Discovery StudioClient v17.2.0.16349. In Discovery Studio Client v17.2.0.16349, theratios of solvent-exposed surface area were calculated by the aboveformula (II).

(3) Result

Table 3 and Tables 4A and 4B show the amino acid frequencies of R, S, T,V, D and E at each position of FR1 and FR3 of a light chain of awild-type anti-lysozyme antibody and their total values, and the ratiosof solvent-exposed surface areas of each amino acid residue.

TABLE 3 Position of FR1 of light chain L1 L2 L3 L4 L5 L6 L7 L8 L9 L10L11 L12 R 0.2 0.1 0.6 0.0 0.1 0.1 0.1 1.0 0.1 0.0 0.1 0.1 S 7.3 16.6 1.80.1 4.6 3.1 49.9 6.0 55.9 58.6 0.4 79.1 T 0.2 1.6 2.4 0.4 88.5 1.4 11.71.7 1.1 23.5 0.7 3.4 V 0.2 9.1 53.4 7.5 0.1 0.0 0.2 0.2 2.1 1.3 29.0 0.3D 37.4 0.3 1.0 0.0 0.3 0.2 1.5 0.1 3.4 0.0 0.0 0.0 E 17.7 0.1 8.6 0.10.1 0.1 2.8 0.2 0.6 0.0 0.2 0.3 Total value of amino acid frequencies(%) 62.9 27.7 67.8 8.2 93.8 4.9 66.2 9.1 63.2 83.5 30.3 83.1 Ratio ofsolvent-exposed surface area (%) 69.0 7.1 88.1 0.0 84.8 0.0 100.0 54.8111.5 74.0 26.8 57.7 Position of FR1 of light chain L13 L14 L15 L16 L17L18 L19 L20 L21 L22 L23 R 0.2 0.1 0.1 0.7 0.3 44.7 0.0 5.7 0.0 0.5 0.1 S1.1 69.3 0.5 0.2 0.9 10.7 0.1 16.0 0.0 47.5 0.1 T 3.5 11.8 0.6 0.0 0.424.3 0.1 70.6 0.1 42.9 0.0 V 31.2 0.3 21.9 0.1 0..0 0.1 61.1 0.2 1.4 0.00.0 D 0.0 0.1 0.0 0.1 25.5 0.0 0.0 0.0 0.0 0.1 0.0 E 1.5 0.0 0.1 0.331.0 0.2 0.0 1.0 0.0 0.2 0.0 Total value of amino acid frequencies (%)37.5 81.6 23.2 1.4 58.1 80.0 61.4 93.5 1.5 91.1 0.2 Ratio ofsolvent-exposed surface area (%) 10.9 67.7 30.6 81.7 85.1 79.9 4.6 81.51.5 25.7 0.0

TABLE 4A Position of FR3 of light chain L57 L58 L59 L60 L61 L62 L63 L64L65 L66 L67 L68 R 0.22 0.00 0.04 0.04 98.50 0.00 0.95 0.03 1.01 3.680.05 0.99 S 0.10 0.07 6.72 27.74 0.13 0.12 88.20 0.73 95.85 3.85 93.332.23 T 0.03 2.07 0.33 0.64 0.17 0.01 5.67 0.02 0.92 1.13 0.25 0.18 V0.06 66.28 0.00 1.06 0.00 0.25 0.77 0.23 0.03 0.87 0.03 0.16 D 2.09 0.070.00 43.09 0.00 0.02 0.01 0.19 0.06 0.05 0.12 0.87 E 0.41 0.03 0.01 7.110.00 0.00 0.02 0.02 0.04 0.35 0.04 0.80 Total value of amino acidfrequencies (%) 2.89 68.52 7.10 79.67 98.81 0.39 95.62 1.20 97.91 9.9393.82 5.22 Ratio of solvent-exposed surface area (%) 123.56 6.65 25.77132.72 13.71 0.34 84.95 2.09 93.83 54.45 85.39 46.07 Position of FR3 oflight chain L69 L70 L71 L72 L73 L74 L75 L76 L77 L78 L79 L80 R 0.69 0.221.33 0.05 0.01 0.96 0.01 0.78 17.70 0.09 2.96 1.01 S 5.49 16.37 0.5826.20 0.09 2.58 0.06 73.92 35.17 0.06 0.07 12.11 T 68.95 18.75 0.1465.26 0.15 74.45 0.17 14.80 1.31 2.22 0.04 8.53 V 0.05 0.44 1.34 0.390.06 0.35 1.72 0.03 0.04 28.04 0.25 1.55 D 2.22 44.95 0.17 0.03 0.000.26 0.00 0.59 1.39 0.01 0.47 0.13 E 0.10 8.77 0.01 0.00 0.01 0.19 0.000.29 0.26 0.01 33.22 1.73 Total value of amino acid frequencies (%)77.50 89.49 3.57 91.94 0.32 78.79 1.96 90.41 55.86 30.44 37.00 25.05Ratio of solvent-exposed surface area (%) 69.13 63.96 0.00 53.09 0.0058.10 0.00 93.56 82.29 0.00 61.80 44.13

TABLE 4B Position of FR3 of light chain L81 L82 L83 L84 L85 L86 L87 L88R 0.03 0.01 0.00 0.01 0.05 0.00 0.01 0.09 S 0.04 0.00 1.00 0.65 1.410.02 0.62 0.05 T 0.17 0.01 0.75 0.44 27.76 0.00 0.01 0.00 V 0.25 0.0611.70 0.66 27.58 0.01 0.05 0.02 D 8.60 99.26 0.74 0.04 32.55 0.00 0.000.00 E 81.18 0.19 34.30 0.01 1.68 0.00 0.00 0.00 Total value of 90.2799.53 48.49 1.81 91.03 0.03 0.69 0.16 amino acid frequencies (%) Ratioof 77.62 1.49 7.43 8.38 15.55 0.00 1.01 0.00 solvent- exposed sur- facearea (%)

From these tables, as a feature common to the variants of Example 1 withimproved affinity for an antigen, it was found that 3 of the amino acidresidues satisfying both following conditions (a) and (b) in the aminoacid sequence of FR of a light chain of an antibody were changed tocharged amino acid residues. It was found that the amino acid residuesmodified by a variant whose expression could not be confirmed in Example1 did not satisfy following conditions (a) and/or (b).

(a) In the amino acid sequence of FR of a light chain of an antibody,present at positions where total value of amino acid frequencies ofarginine, serine, threonine, valine, aspartic acid and glutamic acid is35% or more.

(b) In the amino acid sequence of FR of a light chain of an antibody,has a ratio of solvent-exposed surface area of 20% or more.

Example 3 Preparation of Antibody in which Amino Acid Residue of FR3 ofLight Chain is Modified

Whether the affinity of the antibody for an antigen could be improved bysubstituting 3 of the amino acid residues satisfying both the aboveconditions (a) and (b) found in Example 2 with charged amino acidresidues was verified. Specifically, a variant of anti-lysozyme antibodydifferent from the variants prepared in Example 1 were prepared, andtheir affinity for an antigen was measured.

(1) Preparation of Variant of Anti-Lysozyme Antibody

Three amino acid residues to be modified in light chain FR3 of awild-type anti-lysozyme antibody were selected from Tables 4A and 4B. Inorder to substitute the 3 selected predetermined amino acid residueswith arginine residues, PCR was performed in the same manner as inExample 1 using primers represented by following base sequences. Primersof SEQ ID NOs: 23 to 28 were used as forward primers, and primers of SEQID NOs: 29 to 34 were used as reverse primers.

[Primer for Preparing Variants]

60, 63, 65 Variant FOR: (SEQ ID NO: 23)5′ GAGGCTCGGGCACCGACTTCACCCTGTC 3′ 60, 76, 77 Variant FOR:(SEQ ID NO: 24) 5′ TCGGGCACCGACTTCACCCTGTCGATCAGAAGAGTCGAGACGGAGGA C 3′65, 67, 70 Variant FOR: (SEQ ID NO: 25)5′ CCAGATTCACCCTGTCGATCAACAGCGTCGAG 3′ 67, 70, 72 Variant FOR:(SEQ ID NO: 26) 5′ CCAGATTCAGACTGTCGATCAACAGCGTCGAGAC 3′74, 76, 77 Variant FOR: (SEQ ID NO: 27) 5′ AGTCGAGACGGAGGACTTCGG 3′77, 79, 81 Variant FOR: (SEQ ID NO: 28)5′ AGAACGAGAGACTTCGGCATGTACTTCTGC 3′ 60, 63, 65 Variant REV:(SEQ ID NO: 29) 5′ TGCCTCTGAAGCGTCTCGGGATCCCCGAGATC 3′60, 76, 77 Variant REV: (SEQ ID NO: 30)5′ GCCCGAGCCGCTGAAGCGTCTCGGGATCCCCGAGATC 3′ 65, 67, 70 Variant REV:(SEQ ID NO: 31) 5′ TGCCTCTGCCTCTGCCGCTGAAGC 3′ 67, 70, 72 Variant REV:(SEQ ID NO: 32) 5′ TGCCTCTGCCCGAGCCGCTGAAG 3′ 74, 76, 77 Variant REV:(SEQ ID NO: 33) 5′ CTTCTGATTCTCAGGGTGAAGTCG 3′  77, 79, 81 Variant REV:(SEQ ID NO: 34) 5′ GACTCTGTTGATCGACAGGGTGAAGTCG 3′

Using the obtained PCR product, plasmids containing a gene encoding alight chain of variant and a plasmid containing a gene encoding awild-type heavy chain were obtained in the same manner as in Example 1.Using these plasmids, each antibody was expressed in Expi293 (trademark)cells, and the resulting culture supernatant was purified in the samemanner as in Example 1 to obtain a solution of variant of anti-lysozymeantibody.

(2) Measurement of Affinity

The affinity of the prepared variants was measured using Biacore(registered trademark) T200 (GE Healthcare) in the same manner as inExample 1. The results are shown in Table 5.

TABLE 5 Anti-lysozyme antibody K_(d) (M) Wild-type 1.3E−10 60, 63, 65Variant 6.6E−11 67, 70, 72 Variant 1.4E−11 74, 76, 77 Variant 5.6E−1177, 79, 81 Variant 1.0E−10 65, 67, 70 Variant 2.0E−11 60, 76, 77 Variant9.7E−11

As shown in Table 5, Kd values of all the variants were lower than theKd value of the wild-type anti-lysozyme antibody. Therefore, it wassuggested that the affinity of the antibody for an antigen can beimproved by substituting 3 of the amino acid residues satisfying boththe above conditions (a) and (b) found in Example 2 with arginineresidues.

Example 4 Preparation of Antibody in which Amino Acid Residue of FR1 orFR3 of Light Chain is Modified (2)

As unmodified antibodies, a mouse anti-lysozyme antibody of clone(Hy-HEL5) different from the anti-lysozyme antibody of Example 1, amouse anti-thyroid stimulating hormone (TSH) antibody, and a humanizedanti-HER2 antibody (trastuzumab) were used. In FR1 or FR3 of lightchains of these antibodies, whether the affinity of the antibody for anantigen can be improved by substituting 3 of the amino acid residuessatisfying both the above conditions (a) and (b) with charged amino acidresidues was verified.

(1) Preparation of Variants (1.1) Obtainment of Gene Encoding LightChain of Variant of Anti-Lysozyme Antibody (Hy-HEL5)

From Table 3, 3rd, 5th and 9th amino acid residues of light chain FR1were selected as the amino acid residues satisfying both the aboveconditions (a) and (b). In order to substitute these amino acid residueswith histidine residues, PCR was performed in the same manner as inExample 1 using primers represented by following base sequences. Aprimer of SEQ ID NO: 46 was used as a forward primer, and a primer ofSEQ ID NO: 47 was used as a reverse primer.

3, 5, 9 Variant (His) FOR: (SEQ ID NO: 46)5′ CATCTGCACCAATCACCGCACATTATGTCCGCATCTC 3′ 3, 5, 9 Variant (His) REV:(SEQ ID NO: 47) 5′ TATGTCCCCTCTGCTCATAATCACAGAGGCACTG 3′

(1.2) Obtainment of Gene Encoding Light Chain of Variant of Anti-TSHAntibody

The total values of amino acid frequencies shown in Table 3 were used astotal values of amino acid frequencies of R, S, T, V, D and E at eachposition of an amino acid sequence of light chain FR of a wild-typeanti-TSH antibody. Ratios of solvent-exposed surface area of each aminoacid residue of the light chain FR of the anti-TSH antibody wereobtained based on three-dimensional structure data of a light chain of awild-type anti-TSH antibody downloaded from the database PDB in the samemanner as in Example 2. 18th, 20th and 22nd Amino acid residues of thelight chain FR1 were selected as the amino acid residues satisfying boththe above conditions (a) and (b). In order to substitute these aminoacid residues with lysine residues, PCR was performed in the same manneras in Example 1 using primers represented by following base sequences. Aprimer of SEQ ID NO: 48 was used as a forward primer, and a primer ofSEQ ID NO: 49 was used as a reverse primer.

18, 20, 22 Variant (Lys) FOR: (SEQ ID NO: 48)5′ AAGGCCAAGATTAAGTGCAGATCTAATCAGAGCGTTG 3′18, 20, 22 Variant (Lys) REV: (SEQ ID NO: 49)5′ ATCTCCAAGACTGACAGGCAGGGAGAGTG 3′

(1.3) Obtainment of Gene Encoding Light Chain of Variant of HumanizedAnti-HER2 Antibody

Amino acid sequences of light chains of about 30,000 human antibodieswere downloaded as reference antibodies from database abYsis. In thesame manner as in Example 2, the obtained amino acid sequences of lightchains of reference antibodies were aligned, and total value of aminoacid frequencies of R, S, T, V, D and E at each position of the aminoacid sequence of FR was calculated. The ratio of solvent-exposed surfacearea of each amino acid residue of the light chain FR of the anti-HER2antibody was obtained based on three-dimensional structure data of alight chain of a humanized anti-HER2 antibody downloaded from thedatabase PDB in the same manner as in Example 2. 76th, 77th and 81stAmino acid residues of the light chain FR3 were selected. In order tosubstitute these amino acid residues with arginine residues, PCR wasperformed in the same manner as in Example 1 using primers representedby following base sequences. A primer of SEQ ID NO: 50 was used as aforward primer, and a primer of SEQ ID NO: 51 was used as a reverseprimer.

76, 77, 81 Variant (Arg) FOR: (SEQ ID NO: 50)5′ CAGCCGAGAGACTTCGCCACGTATTACTG 3′ 76, 77, 81 Variant (Arg) REV:(SEQ ID NO: 51) 5′ CAGTCTTCTGATCGTCAGGGTAAAATCGGTAC 3′

(1.4) Obtainment of Variants of Each Antibody

Using the obtained PCR product, plasmids containing a gene encoding alight chain of variant of each antibody and a plasmid containing a geneencoding a wild-type heavy chain of each antibody were obtained in thesame manner as in Example 1. Using these plasmids, each antibody wasexpressed in Expi293 (trademark) cells, and the resulting culturesupernatant was purified in the same manner as in Example 1 to obtain asolution of variant of each antibody. An amino acid sequence of a lightchain of 76, 77, 81 variant (Arg) of the humanized anti-HER2 antibody isshown in SEQ ID NO: 52.

(2) Measurement of Affinity

The affinity of the prepared variants was measured using Biacore(registered trademark) T200 (GE Healthcare) in the same manner as inExample 1. In the measurement, TSH protein (R&D Systems, Inc.) was usedas an antigen of the anti-TSH antibody, and HER2 protein (R&D Systems,Inc., Catalog No. 1129-ER) was used as an antigen of the anti-HER2antibody. The results are shown in Table 6. In the table, “Ratio” is aratio of Kd value of variant type when Kd value of wild-type is 1.

TABLE 6 K_(d) (M) Ratio Anti-lysozyme antibody Wild-type 1.77E−10 1.003, 5, 9 Variant (His) 1.21E−10 1.46 Anti-TSH antibody Wild-type 1.18E−091.00 18, 20, 22 Variant (Lys) 4.64E−10 2.55 Anti-HER2 antibody Wild-type5.94E−11 1.00 76, 77, 81 Variant (Arg) 2.19E−11 2.71

As shown in Table 6, Kd values of all the variants were lower than Kdvalues of the wild-type antibodies. Therefore, even for an antibodydifferent from the anti-lysozyme antibody of Example 1, it was suggestedthat the affinity of the antibody for an antigen can be improved bymodifying 3 of the amino acid residues satisfying both the aboveconditions (a) and (b).

What is claimed is:
 1. A method for improving affinity of an antibodyfor an antigen, comprising, in an unmodified antibody, improvingaffinity for an antigen as compared to the unmodified antibody, bychanging 17th, 18th and 20th amino acid residues of a light chaindefined by Kabat method to charged amino acid residues.
 2. The methodaccording to claim 1, wherein the charged amino acid residue is a basicamino acid residue.
 3. The method according to claim 2, wherein thebasic amino acid residue is an arginine residue or a lysine residue. 4.The method according to claim 1, wherein the antibody is an antibodyfragment.
 5. The method according to claim 4, wherein the antibodyfragment is a Fab fragment, a F(ab′)2 fragment, a Fab′ fragment, a Fdfragment, a Fv fragment, a dAb fragment, scFv, or rIgG.
 6. A method forproducing an antibody with improved affinity for an antigen as comparedto an unmodified antibody, comprising: in an unmodified antibody,changing 17th, 18th and 20th amino acid residues of a light chaindefined by Kabat method to charged amino acid residues; and recoveringthe antibody obtained in the changing.
 7. The method according to claim6, wherein the charged amino acid residue is a basic amino acid residue.8. The method according to claim 7, wherein the basic amino acid residueis an arginine residue or a lysine residue.
 9. The method according toclaim 6, wherein the antibody is an antibody fragment.
 10. The methodaccording to claim 9, wherein the antibody fragment is a Fab fragment, aF(ab′)2 fragment, a Fab′ fragment, a Fd fragment, a Fv fragment, a dAbfragment, scFv, or rIgG.
 11. A modified antibody with improved affinityfor an antigen as compared to an unmodified antibody, wherein 17th, 18thand 20th amino acid residues of a light chain defined by Kabat method inthe unmodified antibody are changed to charged amino acid residues. 12.The modified antibody according to claim 11, wherein the charged aminoacid residue is a basic amino acid residue.
 13. The modified antibodyaccording to claim 12, wherein the basic amino acid residue is anarginine residue or a lysine residue.
 14. The modified antibodyaccording to claim 11, which is an antibody fragment.
 15. The modifiedantibody according to claim 14, wherein the antibody fragment is a Fabfragment, a F(ab′)2 fragment, a Fab′ fragment, a Fd fragment, a Fvfragment, a dAb fragment, scFv, or rIgG.